There exists in the prior art a body of references which disclose the use of certain esters which, when cleaved by enzymatic activity, result in the formation of color or other detectable species. Thus, British Pat. No. 1,128,371 discloses the use of indoxyl and thioindoxyl esters as useful chromogens in detecting hydrolytic enzymes in body fluids. The enzymes cleave the ester to generate free indoxyl, which subsequently oxidizes to form the dimeric product indigo, a readily observable blue dye. Such activity is said to be due to, among other enzymes, cholinesterase. This patent also teaches that, in addition to the indoxyl portion of the ester substrate, the acid radical is chosen with particular reference to the enzyme to be detected. For example, it is stated that the acid radical can be acetate, or laurate or stearate for detection of esterase or lipase, respectively. For detecting enzymes such as phosphatase or sulfatase the acyl radical can be inorganic. Thus, the British Patent can be held to teach the use of chromogenic esters as substrates for determining esterolytic enzymes, such esters comprising indoxyl or thioindoxyl as the alcoholic moiety of the ester, the acyl moiety being tailored to be conducive to the particular enzyme to be determined.
The effect of careful acyl radical selection is nowhere more clearly exemplified than in two references which demonstrate esterase specificity for esters in which the acyl radical comprises an N-protected amino acid or peptide. Thus Janoff, et al., Proc. Soc. Exper. Biol. Med. 136: 1045-1049 (1971) teaches that alanine esters are specific substrates for esterase obtained from human leukocytes. Specifically this reference teaches that an extract of human leukocyte granules is capable of hydrolyzing N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester. Moreover, L-alanine-p-nitrophenol ester was similarly hydrolyzed to yield the yellow p-nitrophenol colorform.
Similarly, Sweetman et al., Jour. Hist. Soc., 22: 327-339 teaches the use of 1-naphthyl-N-acetyl-DL-alanine and 1-naphthyl butyrate to demonstrate the presence of esterase, as well as 1-naphthyl-N-acetyl-L-alanyl-L-alanine.
U.S. Pat. No. 4,278,763, assigned to Boehringer Mannheim GmbH combines these teachings in arriving at the indoxyl or thioindoxyl esters of amino acids or peptides as still another example of a traditional chromogenic substrate for leukocytic esterase activity. Moreover the Boehringer patent teaches the equivalence of proteases and esterase in their esterolytic penchants.
It is known that ester hydrolysis reactions can be activated by the presence of many nucleophilic agents, including many alcohols. Thus, the rate of hydrolysis of phenyl acetate and p-nitrophenyl acetate by esterase is increased 2.5 to 5.5 times upon addition of methanol and butanol. Greenzaid and Jencks, Biochemistry, 10(7), 12101227 (1971). Moreover, the effect increases with the length of the n-alkyl group. Wynne and Shalatin, Eur. J. Biochem., 31, 554-560 (1972).
In particular, this activation effect of alcohols has been observed with esters of amino acids. p-Nitrophenyl N-acetyl-L-alaninate hydrolysis is activated (accelerated) by the presence of methanol. Fastrez and Fersht, Biochemistry, 12(11), 2025-2034 (1973). High molecular weight alcohols increase the rate of esterase-induced hydrolysis of p-nitrophenyl t-BOC-L-tyrosinate. Ashe and Zimmer, Biochem. and Biophys. Res. Comm., 75(1), 194-199 (1977). The disclosure of U.S. Pat. No. 4,299,917 describes other known ester hydrolysis activators such as certain metal complexes, pyramine derivatives and imidazoles.
Also known is the use of certain diazonium salts to couple with phenols and pseudophenols to produce azo dyes. Martinet and Dornier Compt. Rend., 170, 592 (1920). Such a technique is used in an esterase analysis whereby indoxyl acetate is hydrolyzed via esterase to produce indoxyl, which is in turn coupled with a diazonium salt to form the corresponding azo dye. Holt and Hicks, J. Cell Biol. 29, 361-366 (1966); Gossrau, Histochemistry, 57, 323-342 (1978); West German Offenlegungschrift No. 31 17 721, filed May 9, 1980.
The dye industry, an art which is clearly not analogous to medical diagnostic assays, but which nevertheless has long been a reservoir of experimental organic chemistry procedures, provides some guidance as to certain amino acids and their cyclization reactions. Thus, it is known to form pyrroles through the addition of glycine to salts of .beta.-phenylglycidic acid, followed by treatment with hot acetic anhydride. Madelung and Obermann, Ber., 63, 2870 (1930).